Methods and compositions for generating pacemaker cells

ABSTRACT

Disclosed herein are methods and compositions for generating pacemaker cells from non-pacemaker cardiomyocytes. For example, the method includes the step of culturing the non-pacemaker cardiomyocytes with silk fibroin so that the silk fibroin induces the transformation of at least a portion thereof into pacemaker cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/798,619, filed Oct. 31, 2017, the entire contents of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure relates to methods and compositions forgenerating the biological pacemakers; more particularly, to methods andcompositions that use silk fibroins for generating the biologicalpacemakers.

2. Description of Related Art

The term “cardiac arrhythmia” encompasses a group of medical conditionsin which the heartbeat is too fast, too slow, or irregular. The symptomsof cardiac arrhythmia range from barely perceptible to cardiovascularcollapse and death.

Cardiac arrhythmias are due to problems with the electrical conductionsystem of the heart. The cardiac conduction system comprises a group ofspecialized cardiac muscle cells that send electrical signals to theheart muscle and thereby cause it to contract. The main components ofthe cardiac conduction system are the sinoatrial node (SAN),atrioventricular (AV) node, bundle of His, and Purkinje fibers. Duringcardiogenesis, cardiomyocytes become specialized to exhibit eitherventricular, atrial, or pacemaker properties. The SAN, the primarypacemaker region of the heart, is a highly-specialized structurecontaining approximately 10,000 to 15,000 pacemaker cells or less. Thesepacemaker cells in the SAN generate an electrical impulse thatoriginates from the right atrium of the heart, in particular, the SAN.This impulse then passes through the AV node and through both ventriclesvia the Bundle of His and the Purkinje fibers. The result is asynchronized contraction of the heart muscle, and thus, blood flow.

Normal heart rates range from 50 to 100 beats per minute in an adult atrest. The term bradyarrhythmias refers to a heart rate slower than thenormal heart rate (i.e., fewer than 50 times per minute).Bradyarrhythmias occur when electrical signals from the cardiacconduction system slow down or are blocked; common causes ofbradyarrhythmias include sinus bradycardia (slow electrical impulsesfrom the sinus node), sinus arrest (pauses in the normal activity of thesinus node), and AV block (blockages of the electrical impulse from theatria to the ventricles). Bradycardia may lead to low cardiac output andoxygen-rich blood in the body, which is associated with exerciseintolerance, syncope, and sudden cardiac death.

Current therapies for cardiac bradyarrhythmias rely on an implantedelectronic pacemaker that helps the heart maintain an appropriate rate.However, the electronic pacemakers are quite expensive, and theimplantation of such electronic devices may lead to variouscomplications, including pulmonary collapse, hemorrhage, bacterialinfection, as well as device malfunctions (e.g., lead/generatorfailure). These drawbacks limit the applicability of this technique.Therefore, the related art seeks eagerly for alternative treatmentoptions of bradyarrhythmias.

Several investigational therapies, including cell therapies and genetherapies, have been reported to create biological pacemakers whichproduce specific electrical stimuli that mimic that of the body'snatural pacemaker cells. In the cell therapy, stem cells (includinginduced pluripotent stem cells or embryonic stem cells) are convertedinto pacemaker cells. In gene therapy, the biological pacemakers aremade by the expression of genes that increase excitability or diastolicdepolarization, or that specify a sinus-node phenotype. Yet the clinicalapplication of these technologies remains obscure. To begin with, theadministration of foreign cells during the cell therapy often triggerthe immune response of the recipient. Further, there is no standardizedway to manufacture and validate the cell products to be used. Also, thebeating rates from implanted cardiomyocytes or transgenic stem cell aretoo slow to fit the needs until now. On the other hand, gene therapyinvolving, for example, TBX18 gene, could create a satisfactory beatingrate. The effect, however, seems short-term. However, the use of viralvectors for the gene therapy might induce the cardiac immune response.Additionally, the manufacture and validation of the viral product arealso difficult to standardize. Vectors containing other genes, includingTBX3, hyperpolarization-activated cyclic nucleotide-gated (HCN)channels, beta-2 receptors, adenylyl cyclase, SKM1 are facing similarproblems.

In view of the foregoing, there exists a need in the related art forproviding a method and composition for generating pacemaker cells so asto address the disadvantages faced by conventional art.

SUMMARY

The following presents a simplified summary of the disclosure in orderto provide a basic understanding to the reader. This summary is not anextensive overview of the disclosure and it does not identifykey/critical elements of the present invention or delineate the scope ofthe present invention. Its sole purpose is to present some conceptsdisclosed herein in a simplified form as a prelude to the more detaileddescription that is presented later.

Considering the facts discussed above, one purpose of the presentinvention is to provide a novel strategy to generate cells withpacemaker activity; in some embodiments, cells with induced pacemakeractivity are similar to those in the SAN. Moreover, the proposedpreparation method is easily standardizable. Therefore, in one aspect,the present disclosure is directed to a method for generating pacemakercells (e.g., SAN-like cells) from non-pacemaker cardiomyocytes.

According to some embodiments, the method comprises the step ofcontacting a cell population comprising non-pacemaker cardiomyocyteswith an effective amount of a silk fibroin biomaterial so that the oneor more of the cells are transformed into the pacemaker cells.

In some optional embodiments, the cell population and the silk fibroinbiomaterial are contacted in vitro. In these cases, the method furthercomprises the step of culturing the cell population with the silkfibroin biomaterial under a suitable condition so that one or more ofthe non-pacemaker cardiomyocytes are transformed into the pacemakercells.

In other optional embodiments, the cell population and the silk fibroinbiomaterial are contacted in vivo so that at least a portion of thenon-pacemaker cardiomyocytes are transformed into the pacemaker cells insitu.

According to various embodiments of the present disclosure, the silkfibroin biomaterial is a silk fibroin solution, a silk fibroin particle,a non-woven silk fibroin mat, a silk fibroin hydrogel, a silk fibroinfilm, or a silk fibroin scaffold. For example, the silk fibroin filmcomprises a biodegradable polymeric film having a plurality of aminesgroup on the surface thereof, and a plurality of silk fibroinscrosslinked with the biodegradable polymeric film via the plurality ofamine groups.

In another aspect, the present invention is directed to pacemaker cells.According to various embodiments of the present disclosure, thepacemaker cells are generated using the in vitro method according to theabove-mentioned aspect/embodiments of the present disclosure. Forexample, the method comprises the step of, culturing a cell populationcomprising non-pacemaker cardiomyocytes with an effective amount of asilk fibroin biomaterial so that the one or more of the non-pacemakercardiomyocytes are transformed into the pacemaker cells.

According to optional embodiments of the present invention, thepacemaker cells comprise SAN-like cells. Still optionally, the SAN-likecells exhibit an increased expression level ofhyperpolarization-activated, cyclic nucleotide-gated potassium channel 4(HCN4) gene or connexin 45 gene, or both, compared with the cells thatare not transformed. Still optionally, the SAN-like cells exhibitsinoatrial node-specific calcium clock.

In yet another aspect, the present disclosure is directed to apharmaceutical composition for treating cardiac arrhythmia.

According to some embodiments, the pharmaceutical composition comprisesan effective number of pacemaker cells according to the above-mentionedaspect/embodiments and a pharmaceutically acceptable carrier thereof.

In optional embodiments, the pharmaceutical composition furthercomprises an effective amount of a silk fibroin biomaterial. Forexample, the silk fibroin biomaterial may be in the form of a silkfibroin solution, a silk fibroin particle, a non-woven silk fibroin mat,a silk fibroin hydrogel, a silk fibroin film, or a silk fibroinscaffold.

In still another aspect, the present disclosure is directed to a methodfor treating a subject suffering from cardiac arrhythmia.

According to certain embodiments, the treatment method comprises thestep of administering to the subject an effective number of pacemakercells or a pharmaceutical composition according to any of theabove-mentioned aspects/embodiments of the present invention.

In some optional embodiments, the cell population used to prepare thepacemaker cells is derived from the subject.

According to some other embodiments, the treatment method involves thein-situ formation of pacemaker cells. In these cases, the methodcomprises the step of administering to the heart of the subject aneffective amount of a silk fibroin biomaterial.

According to various embodiments of the present disclosure, the silkfibroin biomaterial is a silk fibroin solution, a silk fibroin particle,a non-woven silk fibroin mat, a silk fibroin hydrogel, a silk fibroinfilm, or a silk fibroin scaffold. For example, the silk fibroin filmcomprises a biodegradable polymeric film having a plurality of aminesgroup on the surface thereof, and a plurality of silk fibroinscrosslinked with the biodegradable polymeric film via the plurality ofamine groups.

Subject matters that are also included in other aspects of the presentdisclosure include the use of a silk fibroin biomaterial in themanufacture of a medicament for use in the treatment of cardiacarrhythmia or in the generation of pacemaker cells, as well as a silkfibroin biomaterial for use in the treatment of cardiac arrhythmia or inthe generation of pacemaker cells. For example, the silk fibroinbiomaterial may be in the form of a silk fibroin solution, a silkfibroin particle, a non-woven silk fibroin mat, a silk fibroin hydrogel,a silk fibroin film, or a silk fibroin scaffold.

Many of the attendant features and advantages of the present disclosurewill becomes better understood with reference to the following detaileddescription considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present description will be better understood from the followingdetailed description read in light of the accompanying drawings, where:

FIG. 1A and FIG. 1B are bar graphs respectively illustrating theincidence of beating cardiomyocytes and the beating rate (beat perminute) in cardiomyocytes, according to one working example of thepresent disclosure;

FIG. 1C and FIG. 1D are bar graphs respectively illustrating the beatingrate in cardiomyocytes treated with SF solution and SF hydrogel,compared with PBS control, according to one working example of thepresent disclosure;

FIG. 2A and FIG. 2B are bar graphs illustrating the relativequantitation of different marker genes in cardiomyocytes, according toone working example of the present disclosure;

FIG. 2C is a western blot for HCN4, according to one working example ofthe present disclosure;

FIG. 2D presents immunostaining images of cardiomyocytes, according toone working example of the present disclosure;

FIG. 3 is a representative diagram illustrating the action potential ofcardiomyocytes over times, according to one working example of thepresent disclosure;

FIG. 4 presents diagrams illustrating the calcium dynamics ofcardiomyocytes, according to one working example of the presentdisclosure;

FIG. 5 is line-scan confocal imaging of cardiomyocytes, according to oneworking example of the present disclosure;

FIG. 6A is a line graph illustrating the effect of ivabradine (aselective HCN4 inhibitor) on the beating rate of the SF-treatedcardiomyocytes and control cardiomyocytes, according to one workingexample of the present disclosure;

FIG. 6B and FIG. 6C respectively present diagrams illustrating theelectrophysiology properties of control and SF-treated cardiomyocytes,according to one working example of the present disclosure;

FIG. 7 provides photographs illustrating the morphology ofcardiomyocytes, according to one working example of the presentdisclosure;

FIG. 8 provides bar graphs illustrating the morphology ofcardiomyocytes, according to one working example of the presentdisclosure;

FIG. 9 presents immunostaining images of cardiomyocytes, according toone working example of the present disclosure;

FIG. 10 presents diagrams illustrating the electrophysiology propertiesof cardiomyocytes, according to one working example of the presentdisclosure;

FIG. 11A presents representative diagrams illustrating theelectrocardiogram (ECG) tracings of rats before and after AVN ablation(control and treated with 2% (w/w) SF hydrogel);

FIG. 11B is an optical mapping of the heart of a representative rateafter AVN ablation, according to one working example of the presentdisclosure;

FIG. 11C presents representative diagrams illustrating the ECG tracingof rats after AVN ablation (treated with 12% (w/w) SF solution),according to one working example of the present disclosure;

FIG. 12 is a bar graphs illustrating the relative quantitation ofdifferent marker genes in cardiomyocytes, according to one workingexample of the present disclosure; and

FIG. 13 is a bar graphs illustrating the beating rate in cardiomyocytes,according to one working example of the present disclosure.

DESCRIPTION

The detailed description provided below in connection with the appendeddrawings is intended as a description of the present examples and is notintended to represent the only forms in which the present example may beconstructed or utilized. The description sets forth the functions of theexample and the sequence of steps for constructing and operating theexample. However, the same or equivalent functions and sequences may beaccomplished by different examples.

For convenience, certain terms employed in the specification, examplesand appended claims are collected here. Unless otherwise defined herein,scientific and technical terminologies employed in the presentdisclosure shall have the meanings that are commonly understood and usedby one of ordinary skill in the art.

Unless otherwise required by context, it will be understood thatsingular terms shall include plural forms of the same and plural termsshall include the singular. Also, as used herein and in the claims, theterms “at least one” and “one or more” have the same meaning and includeone, two, three, or more. Furthermore, the phrases “at least one of A,B, and C”, “at least one of A, B, or C” and “at least one of A, B and/orC,” as use throughout this specification and the appended claims, areintended to cover A alone, B alone, C alone, A and B together, B and Ctogether, A and C together, as well as A, B, and C together.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in therespective testing measurements. Also, as used herein, the term “about”generally means within 10%, 5%, 1%, or 0.5% of a given value or range.Alternatively, the term “about” means within an acceptable standarderror of the mean when considered by one of ordinary skill in the art.Other than in the operating/working examples, or unless otherwiseexpressly specified, all of the numerical ranges, amounts, values andpercentages such as those for quantities of materials, durations oftimes, temperatures, operating conditions, ratios of amounts, and thelikes thereof disclosed herein should be understood as modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the present disclosureand attached claims are approximations that can vary as desired. At thevery least, each numerical parameter should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques. Ranges can be expressed herein as from oneendpoint to another endpoint or between two endpoints. All rangesdisclosed herein are inclusive of the endpoints, unless specifiedotherwise.

According to the present disclosure, the term “pacemaker cells” refersto specialized cardiomyocytes capable of self-exciting with regularperiodicity. The natural pacemaker cells make up the cardiac pacemaker(i.e., the natural pacemaker) of the heart and directly control theheart rate. Generally, in a normal heart, cardiomyocytes within thesinoatrial (SA) node display the fastest self-excitatory periodicity andthus serve as the sole pacemaker cells. On the other hand,cardiomyocytes within the atrioventricular (AV) node exhibit the secondfastest self-excitatory periodicity and usually arises as the primarypacemaker when the SA node is damaged. Cardiomyocytes within the bundleof His show the next fastest self-excitatory periodicity followed by theremainder of the Purkinje Fibers throughout the ventricle; and hence,cells at these loci emerge as the pacemakers in the event of the SA andAV node damage. Therefore, natural pacemaker cells includecardiomyocytes within both the SA and AV nodes, as well as those withinPurkinje Fibers (including the bundle of His). Moreover, in the presentdisclosure, the term “pacemaker cells” is also referred to cells thatare artificially induced to have the pacemaker activity using themethods proposed herein. On the other hand, the term “non-pacemakercardiomyocytes” as used throughout the present disclosure are cardiacmuscle cells that exhibit no self-excitatory behavior in their naturallocus within the heart. For examples, the cardiomyocytes making up theatria and the ventricles are non-pacemaker cardiomyocytes.

Regarding the term “sinoatrial node-like (or SAN-like) cells,” it isused herein to designate cells artificially derived from non-pacemakercardiomyocytes. For example, the cells may be obtained fromnon-pacemaker cardiomyocytes by the method disclosed in the presentdisclosure. These SAN-like cells are capable of self-exciting. Inpreferred, optional embodiments, the SAN-like cells express one or morecell markers of SAN cells that are generally expressed in a low level ornot expressed in the non-pacemaker cardiomyocytes. Still optionally, oneor more characteristics of the self-excitation pattern of these SAN-likecells are similar or equivalent to that of the normal SAN cells.

As used herein, the term “silk fibroin” or “fibroin” includes silkwormfibroins and silk proteins from arthropods (e.g., spiders, scorpions,and mites) or insects (e.g., bees), or genetically engineered silks(such as silks from bacteria, yeast, mammalian cells, transgenicanimals, and transgenic plants). Silk consists of the fibroin andsericin, in which the fibroin serves as the structural center of thesilk with the sericin being the gum coating the fibers, thereby stickingthem with each other. For example, silk from silkworms (Bombyx mori)consists of about 70 to 80% fibroin and 20 to 30% sericin, and currentlyis the most common source of silk fibroin. Silk fibroin can be attainedby removing the sericin from the silk; a process known as degumming.Examples of conventional degumming processes include boiling the silksin an aqueous solution containing an alkaline sodium salt such as sodiumcarbonate or sodium bicarbonate, immersing the silks in pressurized hotwater (ex. hot water of 120° C.), and enzymatic degumming. The degummedfibers may be further processed into various silk fibroin biomaterials.For example, the degummed fibers may be dissolved to give an aqueoussilk fibroin solution. When further processed together with one or moresuitable biocompatible polymers, the silk fibroin solution may be madeinto non-woven silk fibroin mats, silk fibroin films, silk fibroinhydrogels, or silk fibroin scaffolds. On the other hand, the degummedfibers may be directly made into silk fibroin cords or non-woven silkfibroin mats. Also, the silk fibroin or the silk fibroin biomaterialscan be chemically modified with various active agents to alter thephysical and/or chemical properties, as well as the functionalities, ofthe silk fibroin or the silk fibroin biomaterials.

The terms “treatment” and “treating” as used herein may refer to apreventative (e.g., prophylactic), curative or palliative measure. Inparticular, the term “treating” as used herein refers to the applicationor administration of the present pacemaker cells, a pharmaceuticalcomposition comprising the same, or a silk fibroin biomaterial to asubject, who has a medical condition (e.g., cardiac arrhythmia), asymptom associated with the medical condition, a disease or disordersecondary to the medical condition, or a predisposition toward themedical condition, with the purpose to partially or completelyalleviate, ameliorate, relieve, delay onset of, inhibit progression of,reduce severity of, and/or reduce incidence of one or more symptoms orfeatures of said particular disease, disorder, and/or condition.Treatment may be administered to a subject who does not exhibit signs ofa disease, disorder, and/or condition, and/or to a subject who exhibitsonly early signs of a disease, disorder and/or condition, for thepurpose of decreasing the risk of developing the pathology associatedwith the disease, disorder and/or condition.

The terms “subject” and “patient” are used interchangeably herein andare intended to mean an animal including the human species that istreatable by the pacemaker cells, pharmaceutical compositions comprisingthe same, silk fibroin biomaterials, and/or methods of the presentinvention. Accordingly, the term “subject” or “patient” comprises anymammal, which may benefit from the present disclosure. The term “mammal”refers to all members of the class Mammalia, including humans, primates,domestic and farm animals, such as rabbit, pig, sheep, and cattle; aswell as zoo, sports or pet animals; and rodents, such as mouse and rat.The term “non-human mammal” refers to all members of the class Mammalisexcept human. In one exemplary embodiment, the patient is a human. Theterm “subject” or “patient” intended to refer to both the male andfemale gender unless one gender is specifically indicated.

The terms “application” and “administration” are used interchangeablyherein to mean the application of the pacemaker cells, pharmaceuticalcompositions, or silk fibroin biomaterials of the present invention to asubject in need of such treatment.

The term “effective amount” as used herein refers to the quantity of anagent (e.g., the present induced pacemaker cell, pharmaceuticalcomposition, or silk fibroin biomaterial) that is sufficient to yield adesired therapeutic response. An effective amount of an agent is notrequired to cure a disease or condition but will provide a treatment fora disease or condition such that the onset of the disease or conditionis delayed, hindered or prevented, or the disease or condition symptomsare ameliorated. The effective amount may be divided into one, two, ormore doses in a suitable form to be administered at one, two or moretimes throughout a designated time period. The specific effective orsufficient amount will vary with such factors as particular conditionbeing treated, the physical condition of the patient (e.g., thepatient's body mass, age, or gender), the type of mammal or animal beingtreated, the duration of the treatment, the nature of concurrent therapy(if any), and the specific formulations employed and the structure ofthe compounds or its derivatives. Effective amount may be expressed inany suitable ways. For pacemaker cells, the effective amount may beexpressed as the total number of cells or cells per volume. As to theeffective amount of the pharmaceutical composition, it may be expressed,for example, as the total mass (e.g., in grams, milligrams, ormicrograms) or volume (e.g., in liters, milliliters, or microliters) ofthe medicament, a ratio of mass of the medicament to body mass (such as,milligrams per kilogram (mg/kg)), or the total number of cells or cellsper volume comprised in the medicament. Regarding the effective amountof the silk fibroin, it may be expressed in grams, milligrams ormicrograms. In the case where the silk fibroin is provided in the formof a solution, hydrogel, or where suitable, the effective amount of thesilk fibroin may be expressed in percentage by weight (wt %),mass-to-volume percentage (% m/v), or percentage by volume (vol %).

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, carrier, solvent or encapsulatingmaterial, involved in carrying or transporting the subject ingredients(e.g., pacemaker cells or silk fibroin) from one organ or portion of thebody, to another organ or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation. The pharmaceutical formulation contains thepacemaker cells of the invention in combination with one or morepharmaceutically acceptable carriers for the pacemaker cells. Thecarrier can, for example, be in the form of a semi-solid or liquiddiluent. These pharmaceutical preparations are a further object of theinvention. Usually, the amount of active ingredients is between 0.1-95%by weight of the preparation. For the clinical use of the methods of thepresent invention, the pharmaceutical composition of the invention isformulated into formulations suitable for the intended route ofadministration, such as via injection. In some examples, thepharmaceutically acceptable carrier comprises the silk fibroinbiomaterials (such as the silk fibroin solution or hydrogel).

The present disclosure is based, at least in part, on the unexpecteddiscovery that the silk fibroin treatment may induce the transformationof non-pacemaker cardiomyocytes into pacemaker cells resembling naturalsinoatrial node (SAN) cells. In view of the foregoing, the presentdisclosure provides methods for generating pacemaker cells from a cellpopulation comprising non-pacemaker cardiomyocytes, by treating the cellpopulation with a silk fibroin biomaterial. Some embodiments of thepresent disclosure are directed to methods for treating disordersassociated with cardiac arrhythmia. Also provided herein is the use ofaforementioned pacemaker cells or silk fibroin biomaterials in thetreatment of cardiac arrhythmia, as well as for use in the manufactureof a medicament for said treatment purpose. The pacemaker cellsgenerated using the proposed method are, of course, the subject mattercovered by the scope of the present application. Further, theabove-mentioned medicament (i.e., a pharmaceutical compositioncomprising the pacemaker cells or silk fibroin biomaterials or both) isalso within the scope of the present application.

The present disclosure is advantageous in at least the following aspect.First, silk fibroins biomaterials are safe for animals, including human,and hence, they are suitable for use not only in vitro but also in vivoand ex vivo. Further, silk fibroins may be made into various forms ofbiomaterials depending on the use and/or application route thereof,which greatly increases the applicability of the present method. Also,the silk fibroin is an abundant resource that is readily accessible witha relatively inexpensive, as compared with stem cell therapies or genetherapies. Moreover, the preparation methods of different silk fibroinsbiomaterials are readily standardizable, providing a stable and reliablemeans for carrying out the present method.

In view of the foregoing, the first aspect of the present disclosure isdirected a method for generating pacemaker cells (or SAN-like cells).

According to some embodiments of the present disclosure, the methodcomprises the step of contacting a cell population comprisingnon-pacemaker cardiomyocytes with an effective amount of a silk fibroinbiomaterial.

As could be appreciated, the present method may be carried out in vitro,in vivo, or ex vivo. In the case where the population of cells and thesilk fibroin biomaterial are contacted in vitro, the method furthercomprises the step of culturing the population of cells with the silkfibroin biomaterial so that one or more of the non-pacemakercardiomyocytes are transformed into the pacemaker cardiomyocytes orSAN-like cells. For instance, the population of cells and the silkfibroin biomaterial are cultured in a medium suitable for the cells, andthen placed under a condition that facilitates the growth, proliferationand/or transformation of the cells. As to the ex vivo culture, thepopulation of cells or the tissue or organ comprising the non-pacemakercardiomyocytes is contacted with the silk fibroin biomaterial under acondition sufficient to transform one or more of the non-pacemakercardiomyocytes into pacemaker cells. For in vivo transformation of thecells, the silk fibroin biomaterial is applied to the target site withina subject's body; in particular, the silk fibroin biomaterial isformulated in a form that increases the retention of the silk fibroinsat the target site, so that a sufficient number of non-pacemakercardiomyocytes at the target site are transformed into pacemaker cellsunder the induction of silk fibroins.

According to various optional embodiments of the present disclosure, theabove-mentioned non-pacemaker cardiomyocytes may be atrial andventricular cardiac muscle cells.

As could be appreciated, these cardiac muscle cells do not generateelectrical impulses (or action potentials) spontaneously in theirnatural locus within the heart, and hence the name “non-pacemakercardiomyocytes.”

The silk fibroin biomaterial may be made into different forms dependingon the setting in which the silk fibroin biomaterial is used, as long asthey allow an adequate contact between the silk fibroin biomaterials andthe cells to be treated. According to some embodiments of the presentdisclosure, aqueous solutions (SF solutions) containing about 0.1 to 50%(w/w) silk fibroins are used. Specifically, the SF solutions may containabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5,26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5,33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5,40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5,47, 47.5, 48, 48.5, 49, 49.5, or 50% (w/w) silk fibroins. Alternatively,silk fibroin particles (SF particles) having an average diameter in therange of about 50 nm to 10 mm are administered to the cells; forexamples, the cells and the SF particles may be mixed in vitro or exvivo. Preferable, the average diameter of SF particles may be 100 nm to5 mm; more preferably, 200 nm to 1 mm, 500 nm to 500 μm, 1 μm to 100 μm,or 10 μm to 50 μm. Still alternatively, the silk fibroin biomaterialsmay be made into non-woven silk fibroin mats (SF non-woven mats) or silkfibroin scaffolds (SF scaffolds); these biomaterials have athree-dimensional structure with an increased surface area, whichfacilitates the retention of cells. For example, the SF non-woven matsor SF scaffolds may be used to culture and transform the cells in vitro,and then, the thus-generated pacemaker cells are collected andadministered to a subject.

Alternatively, the pacemaker cells are administered to the subject alongwith the silk fibroin biomaterials. As another example, the SF non-wovenmats or scaffolds may be administered to the target site of the subjectand then induce the transformation of non-pacemaker cardiomyocytes intopacemaker cells in situ. Likewise, silk fibroin hydrogels (SF hydrogels)and silk fibroin films (SF films) also promote the retention of cells,as well as prolong the residence of the biomaterials at the target site.Accordingly, SF hydrogels or SF films may be used for in vitro or exvivo culture or in vivo induction, or a combination thereof.

According to certain embodiments of the present disclosure, a silkfibroin film is provided. The SF film comprises a biodegradablepolymeric film, which is chemically modified to increase the aminesgroup on the surface thereof, and a plurality of silk fibroinscrosslinked with the biodegradable polymeric film via the amine groups.As could be appreciated, a thin film of silk fibroin may also be formedby coating an SF solution or SF hydrogel (or a liquid or gel containingSF particles dispersed therein) on to the surface of a suitable article(such as, a glass slide, a plastic or metal (e.g., stainless steel)sheet, or a syringe needle), and then allowing the continuous phase toevaporate, thereby obtaining the SF film.

In another aspect, the present invention is directed to pacemaker cellsgenerated using any of the methods described above. According tooptional embodiments of the present invention, the pacemaker cells areSAN-like cells that exhibit an increased expression level of HCN4 geneor connexin 45 gene, or both, compared with the cells that are nottransformed. Still optionally, the SAN-like cells exhibit sinoatrialnode-specific calcium clock. Also, according to certain embodiments, themorphology of the SAN-like cells is similar to that of the naturalpacemaker cells.

In yet another aspect, the present disclosure is directed to apharmaceutical composition for treating cardiac arrhythmia in a subjectin need of such treatment. According to certain embodiments of thepresent disclosure, the pharmaceutical composition comprises aneffective number of pacemaker cells and a pharmaceutically acceptablecarrier for the pacemaker cells. In optional embodiments, thepharmaceutical composition further comprises an effective amount of asilk fibroin biomaterial. For example, the silk fibroin biomaterial maybe in the form of a silk fibroin solution, a silk fibroin particle, anon-woven silk fibroin mat, a silk fibroin hydrogel, a silk fibroinfilm, or a silk fibroin scaffold. According to certain embodiments, thepacemaker cells and the silk fibroin biomaterial may be formulated intoa single composition or separately into two compositions.

According to various embodiments of the present disclosure, thepharmaceutical composition may be formulated into a dosage form suitablefor the desired mode of administration. As could be appreciated, thepacemaker cells can be administered into any area of the heart whereconduction disturbances have occurred. The number of pacemaker cellsnecessary to be therapeutically effective varies with the type ofdisorder being treated as well as the extent of the overall damage ofmyocardial tissue, among other factors. A particularly suitableadministration mode can be in situ application of the present pacemakercells or pharmaceutical composition to a cardiac tissue by, for example,direct surgical application. Another particularly suitableadministration mode can be catheter injection of the present pacemakercells or pharmaceutical composition to a cardiac tissue. There have beenmany known techniques that can be used to facilitate the access to theadministration site. For example, the catheter injection may be used inconnection with a fluoroscopy, X-ray, echocardiography, or magneticresonance imaging guiding system. It should be noted that theabove-mentioned administration routes are provided for the purpose ofdiscussion, and the present disclosure is not limited to theseadministration modes. Rather, according to some embodiments, thepacemaker cells, silk fibroin biomaterials, or pharmaceuticalcompositions may be administered systemically. Illustrative examples ofdosage forms of the pharmaceutical composition include suspensions,dispersions, solutions, ointments, pastes, powders, dressings, creams,and gels. As could be appreciated, these pharmaceutical compositions arealso within the scope of the present disclosure.

In still another aspect, the present disclosure is directed to a methodfor treating a subject suffering from cardiac arrhythmia.

In some embodiments, the treatment method comprises the step ofadministering to the subject an effective number of the presentpacemaker cells or a pharmaceutical composition comprising the same.Alternatively, the treatment method comprises the step of administeringto the subject an effective number of silk fibroin biomaterial or apharmaceutical composition comprising the same, so that the silk fibroinbiomaterial induces the formation of pacemaker cells from non-pacemakercardiomyocytes within the heart of the subject. Still alternatively, thetreatment method comprises the step of administering to the subject aneffective number of the present pacemaker cells and the silk fibroinbiomaterial to the subject. As could be appreciated, the pacemaker cellsand the silk fibroin biomaterial may be formulated in a singlepharmaceutical composition or two separate pharmaceutical compositions;in the latter case, the two pharmaceutical compositions may beadministered to the subject simultaneously or separately.

In some embodiments, about 1 μl to 500 μl of SF hydrogel (prepared usinga 0.1-50% (w/w) SF solution) is administered to a rat subject;optionally, the SF hydrogel may be administered to a rat at a dose ofabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 250, 300, 350, 400, 450, or 500 μl. According to optionalembodiments of the present disclosure, in the case where the subjectsare humans, about 100 μl to 250 ml of SF hydrogel (prepared using a0.1-50% (w/w) SF solution) may be administered. For humanadministration, the dose of the SF hydrogel may be about 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, or 950 or 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 ml.

As to the SF solution (0.1-50% (w/w)), about 10 to 500 μl of SF solutionis administered to the rat; for example, the rat dose of the SF solutionmay be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, or 500 μl. Regarding the human subject, the dose of theSF solution (0.1-50% (w/w)) may be in the range of 1 to 250 ml, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250ml. According to various embodiments of the present disclosure, theconcentration of the SF solution is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,35, 40, 45 or 50% (w/w).

As to the silk fibroin biomaterials in other forms, the dose of thesesilk fibroin biomaterials may be estimated from the silk fibroin contentin the above-mentioned dose of the SF solution or SF hydrogel.Generally, the content of the silk fibroin in other forms of silkfibroin biomaterials may be equivalent as or similar to the silk fibroincontent in the SF solution or SF hydrogel.

In the case where the pacemaker cells are administered to a humansubject, the effective amount is about 100 to 1,000,000 viable pacemakercells; preferable, about 1,000 to 100,000 viable pacemaker cells.

The above-mentioned suitable doses for experimental animals aredetermined from the experimental data provided herein, whereas theequivalent doses for human subjects can be decided by considering thedifferences in the weight, volume, and/or surface area of the heartsbetween the experimental animal and a human subject. As could beappreciated, the above-mentioned effective amount may be administered ina single dose or split into several doses that are administered over asuitable time period at desired time interval.

According to some preferred, optional embodiments of the presentdisclosure, the population of non-pacemaker cardiomyocytes used togenerate the pacemaker cells is derived from the subject to be treated.However, the present invention is not limited thereto.

Also include in the scope of the presently claimed invention are use ofa silk fibroin biomaterial or pacemaker cells for the manufacture of amedicament that can be used in treating cardiac arrhythmia.

The following Examples are provided to elucidate certain aspects of thepresent invention and to aid those of skilled in the art in practicingthis invention. These examples are in no way to be considered to limitthe scope of the invention in any manner. Without further elaboration,it is believed that one skilled in the art can, based on the descriptionherein, utilize the present invention to its fullest extent.

Materials and Methods

(1) Preparation of Silk Fibroin Solution

Briefly, Bombyx mori cocoons were cut into small pieces and boiled in a0.02 M aqueous solution of sodium carbonate (Na₂CO₃) for 90 minutes toremove sericin. The resulting fiber bundles were rinsed with deionizedwater and air-dried at room temperature. The dried fiber bundles werethen mixed with a 9.3 M aqueous solution of lithium bromide (LiBr) in anadequate amount, the reaction mixture is reacted under about 70 to 80°C. for 1 hour to prepare a 20% (w/w) silk fibroin (SF)/LiBr solution.The resulting SF/LiBr solution was then centrifuged (approximately 4,500to 8,000 G for around 10 minutes) and filtered via microfiltration toremove any residual impurities. The SF/LiBr solution was then dialyzedin ddH₂O using a cellulose dialysis tubing with a molecular weightcutoff of 6,000 to 8,000 Da for 3 days. When the pH of the solutionwithin the dialysis tubing was less than 7, it was determined that thesolution was free of LiBr, thereby obtaining the silk fibroin (SF)solution, which was stored at 4° C. before use. The gravimetricmeasurement indicated that the concentration of silk fibroin in thethus-obtained fresh SF solution was about 3-4% (w/w).

(2) Preparation of Silk Fibroin Film

Poly(ε-caprolactone) (PCL; molecular weight 80 kDa, from Sigma, St.Louis, Mo., USA) was dissolved in tetrahydrofuran (THF) at aconcentration of 20%. The PCL/THF solution was casted on a glasscoverslip, evaporated, and a PCL film was formed using aspinning-coating machine (1,000 to 2,000 G for 1 minutes), therebyforming the PCL film with a thickness of about 3 to 10 μm. The dried PCLfilm was then treated with poly(ethylamine) (molecular weight: 800 Da,Sigma, St. Louis, Mo., USA) to increase the amount of surface aminogroups on the PCL, followed by cross-linking a thin layer of 1% SFsolution with 1% glutaraldehyde for several minutes. After air-dried atthe room temperature, the SF films with 20% to 40% crystallinity werefirst prepared. To prepare SF films with more than 40% crystallinity,the SF film with 20% to 40% crystallinity was dipped into 95 to 100%ethanol for several minutes to induce β-sheet formation. To assure thesuccessfully grafting of SF onto the PCL surfaces, photochemicalreactions were performed on the surface of the SF film. Briefly, 200 μlof N-succinimidyl-6-[4′-azido-2′-nitrophenyl-amino]-hexanoate (SANPAH;molecular weight: 492.4 Da, Pierce Chemical) in ethanol solution wasgently added onto a SF film, which was placed in a dark room for severalhours to allow the formation of produce azido-derivative reactants, andthe surface was then irradiated with 60 W UV light (290 to 370 nm) for 3to 5 minutes.

(3) Preparation of Silk Fibroin Hydrogel

Horseradish peroxidase (HRP) (type VI, lyophilized powder; fromSigma-Aldrich, St. Louis, Mo.) was mixed with deionized water to form astock solution with a concentration of 1,000 U/mL. The HRP solution wasthen added to the above-mentioned SF solution in a ratio of 10-20 Unitsof HRP to 1 mL of the SF solution. To initiate the gelation, 0.1-0.2 μlof 30% hydrogen peroxide (Sigma Aldrich, St. Louis, Mo.) solution wasadded into 1 mL of the HRP-SF solution (final concentration of hydrogenperoxide: about 0.003 to 0.006%), and mixed by gentle pipetting prior tosetting.

(4) Isolation and Seeding of Cardiomyocytes

Neonatal rat ventricular myocytes (NRVMs) were isolated from neonatalrat (1- to 2-day old) pups. The pups were decapitated first, and thendipped into 70% ethanol. To isolate the cardiomyocytes, the rib cage ofpups was opened and the heart was exposed. The cut-off ventricle placedin HBSS buffer on ice was then cut into smaller pieces and incubatedwith 0.25% trypsin/HBSS in the flask overnight on shaker at 4° C. Afteraspirating the trypsin, the medium was added into the flask andincubated for 20 minutes at 37° C., the medium was then removed andreplaced with collagen. The supernatant was collected and centrifuged at50G for about 10 minutes. To remove the un-isolated tissue, the cellsuspension was filtered with a 70-μm strainer and centrifuged at 50G for5 minutes, and then re-suspended in the medium. The cell suspension wasplaced into a T175-flask and incubated at 37° C. for 2 hours to allowthe un-wanted fibroblast to adhere. The cells were collected and theNRVMs were seeded onto the fibronectin-coated cover slide or the SF filmat a proper cell density.

(5) Calcium Imaging by Confocal Microscopy

Seeded cardiomyocytes in Tyrode's solution were loaded with Ca′indicators (10 μM Fluo-3/AM for cardiomyocytes; Calbiochem, San Diego,Calif., USA) and incubated at room temperature for 30 minutes in thedark. Fluorescence imaging was performed with a laser scanning confocalmicroscope (Olympus IX71, Olympus America). The fluorescence density (F)was normalized against the baseline fluorescence (F₀) to determine thetransient [Ca²⁺]_(i) changes, which had excluded variations in thefluorescence intensity because of different volumes of injected dye.Offline analysis was performed using Olympus IX71 and ImageJ.

(6) Immunostaining of Cardiomyocytes

Cardiomyocytes were fixed with 4% paraformaldehyde and permeabilizedwith 0.1% Triton-X 100 and then incubated with the appropriate primaryantibody: sarcomeric α-actinin (Sigma-Aldrich), HCN4 (Alomone), beta1adrenergic receptor (Abcam), muscarinic acetylcholine receptor 2 (Abcam)and Alexa Fluor-conjugated secondary antibodies (Invitrogen).Morphometric assays were performed with ImageJ by measuring the celllength (short and long-axis) of each cardiomyocytes.

(7) Real-Time Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from seeded cardiomyocytes using an RNeasy® MiniKit (QIAGEN, Venlo, Netherlands). cDNA was synthesized using aSuperScript® III First-Strand Synthesis System (Invitrogen, Carlsbad,Calif., USA). Real-time quantitative PCR was performed using a RocheLightCycler® 480 Real Time PCR system with a Tagman Master Mix reagent.The primers for the genes of interest (HCN4, Rn00572232_m1; Cx45 (Gjc1),Rn01750705_m1; ANP (Nppa), Rn00561661_m1; Myl2, Rn01480558_g1; NKx2.5,Rn00586428_m1; Acta1, Rn01426628_g1; Tbx18, Rn01445129_m1; Shox2Rn00564672_m1; GAPDH, Rn01775763_g1) were from Invitrogen (Carlsbad,Calif., USA).

(8) Patch Clamp Analysis

Spontaneous action potentials and currents of cardiomyocytes wererecorded at 37° C. in the perforated patch configuration using a AxonCNS 700B amplifier (Molecular Devices, CA, USA) and the pClamp software(version 10; Molecular Devices). The offline data analysis was conductedwith Clampfit software (Molecular Devices, CA, USA) or by means ofOrigin 6.0 software (Microcal, Northampton, USA). Patch pipettes weredrawn from borosilicate glass and heat-polished, and had a resistance of2-5 MQ on filling with intracellular solution which contained 10 mMNaCl, 130 mM potassium aspartate, 0.04 mM CaCl₂, 3 mM Mg-ATP, 10 mMHEPES and 200 μg/ml amphotericin B, pH adjusted to 7.2 with KOH. Theextracellular (bath) solution contained 140 mM NaCl, 5.4 mM KCl, 1 mMMgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, 5.5 mM glucose; the pH was adjusted to7.4 with NaOH. Action potentials (APs) were recorded with a 10-kHzsampling rate.

(9) Animals

Rats were supplied by National Laboratory Animal Center (Taiwan). Allanimals used in working examples of the present disclosure were housedin an animal room under temperature control (24-25° C.) and 12:12light-dark cycle. Standard laboratory chow and tap water were availablead libitum. The experiments procedures were approved by theInstitutional Review Board of Taipei Veterans General Hospital (Taipei,Taiwan) and were performed in compliance with national animal welfareregulations.

(10) Statistical Analysis

Data were analyzed for mean, standard deviation (s.d.), and standarderror of the mean (s.e.m. or SEM). The quantitative figures in this workrepresent the mean±SEM. Unless indicated otherwise, data sets werestatistically evaluated using a two-tailed t test, and confidence levelof P<0.05 was considered significant.

Example 1

SF Increases Automaticity in Neonatal Cardiomyocytes

The NRVMs (5×10⁴ cells) were seeded on coverslips coated withfibronectin (control) or the above-mentioned SF film. Three to five daysafter seeding, the number of spontaneously beating cardiomyocytes werecounted, and incidence of beating cardiomyocytes was calculated asfollows: (number of beating cardiomyocytes)/(total number ofcardiomyocytes)*100%.

The results, as summarized in FIG. 1A, indicated that the number ofspontaneously beating NRVMs on the present SF film was greater than thatin control groups, and the difference was statistically significant (SF,n=184; control, n=239; *P<0.001; 5 independent experiments). Also, asshown in FIG. 1B, the beating rate of beating foci in SF-treated groupswas higher than that in control groups (SF, n=50; control, n=51;*P<0.001; 5 independent experiments).

NRVMs (5×10⁴ cells) were also treated with the SF solution (2-5% (w/w))or PBS (control). Two to three days after the treatment, the mean heartrate (bpm) was measured. The results, as summarized in FIG. 1C indicatedthat in the control group, the mean heart rate was 2.0±2.0 bpm, whereasin the cells treated with the SF solution, the mean heart rate was76.0±31.3 bpm.

For SF hydrogel, NRVMs (5×10⁴ cells) were treated with the SF hydrogelprepared using 2-3% (w/w) SF solution or PBS (control). Three to fivedays after the culturing, the mean heart rate (bpm) was measured. Theresults, as summarized in FIG. 1D indicated that in the control group,the mean heart rate was 49.6±6.0 bpm, whereas in the cells treated withthe SF hydrogel, the mean heart rate was 149.1±16.0 bpm (P<0.05).

These data established that the present SF-treatment was effective ininducing the transformation of ventricular cardiomyocytes into pacemakercells that beat spontaneously.

Example 2

Distinguished Transcriptional Gene Signatures of SAN Cardiomyocytes inSF-Treated Cardiomyocytes

It is well-known that many types of cells exhibit a gene signature inwhich certain unique genes are expressed in higher levels, as comparedwith those of the same genes in other types of cells. For example, HCN4and Connexin 45 (Cx45) genes, among others, are SAN-specific geneshighly expressed in mature SAN cells. On the other hand, ANP and Myl2genes are markers of ventricular cardiomyocytes. Also, there are somecardiac-specific markers (e.g., ACTA1 and NKX2.5) that are expressed incardiomyocytes with de-differentiation. Therefore, in this workingexample, mRNA levels of the aforementioned genes in SF-treatedcardiomyocytes (from Example 1) were determined to investigate whetherthe genetic expression profile of SF-treated cardiomyocytes bearresemblance to that of the native mature SAN cells, or whether theyretain the genetic expression profile of ventricular cardiomyocytes.

As could be seen in FIG. 2A, mRNA expression levels of HCN4 and Cx45 inSF-treated cardiomyocytes were statistically higher than those of thecontrol groups. On the other hand, the mRNA expression levels of ANP andMyl2 genes were significantly lower in SF-treated cardiomyocytes.Specifically, the SF-treated cardiomyocytes exhibited a 3-fold increasein HCN4 mRNA level and 2-fold increase in Cx45 mRNA level, while themRNA levels of ANP and Myl2 in SF-treated cardiomyocytes decreased morethan 3 folds (n=7-12, *P<0.05). In contrast, the expression of Acta1 andNkx2.5 in SF-treated cardiomyocytes were not significantly differentfrom those in control cardiomyocytes (FIG. 2B, P=0.76 for Acta1, andP=0.77 for Nkx2.5, n=7). This indicated the automaticity in SF-treatedcells is not associated with de-differentiation.

The expression of HCN4 in SF-treated cardiomyocytes was furtherconfirmed by western blotting (FIG. 2C) and immunostaining (FIG. 2D).The representative western blot in FIG. 2C indicated the increasedexpression of HCN4 was seen in SF-treated cardiomyocytes, as comparedwith that of the control cells. As can be seen in representativeimmunostaining photographs in FIG. 2D, significant HCN4 expression(green fluorescence) was found in SF-treated cardiomyocytes (panels Aand C), whereas the level of HCN4 was not detectable in control cells(panels D and E). On the other hand, a decrease in sarcomeric α-actinin(α-sm) (red fluorescence) expression was observed in SF-treatedcardiomyocytes (panels B and C), as compared with that of the controlcells (panels E and F). Further, the morphological examination revealedthat SF-treated cardiomyocytes had an elongated or spindleconfiguration, as well as myofibrillar disorganization (FIG. 2D, panelsA to C); which features resembled the morphology of native SAN cells. Onthe other hand, control cells retained the morphology of ventricularcardiomyocytes with organized myofibrils (FIG. 2D, panels D to E). Theblue fluorescence in the immunostaining photographs are stained by DAPI.

Experimental data in this working example evidenced that the SFtreatment effectively transformed cardiomyocytes into SAN-like cells; inparticular, the transformed cells were similar to native SAN cells interms of their gene expression profile and morphology.

Example 3

SF-Treated Cardiomyocytes Exhibit Sinoatrial Node Action Potential

This working example used the patch clamp analysis to elucidate theelectrophysiology of SF-treated cells.

The result indicated that some of the non-pacemaker cardiomyocytestreated with SF (from 4 independent experiments) exhibited spontaneousaction potentials. FIG. 3 is a representative diagram illustrating theaction potential of one SF-treated cardiomyocyte over time, in which theaction potential pattern exhibited a prominent phase 4 depolarizationwith the rest membrane potential set at about −60 mV, which resembledthat of SAN cells. Also, the waveforms of SF-treated cardiomyocytesmimicked those of sinoatrial node. In contrast, cardiomyocytes on thecontrol film required pacing to create an action potential, and noSAN-like action potential was noted (data not shown).

Example 4

SF-Treated Cardiomyocytes Exhibit Sinoatrial Node-Specific Calcium Clock

Automaticity in the SAN cell is the integration between the cyclicdepolarization of action potentials and the intracellular Ca²⁺ cycling.A distinct localized, sub-sarcolemmal Ca²⁺ release (LCR) during thediastolic depolarization (DD) of the SAN cell is another hallmark ofautomaticity in SAN cells, in which the LCR augments the depolarizationrate of the phase 4 action potential and is closely linked with thespontaneous cycle length. Therefore, in this example, calcium imagingwas performed to investigate whether the SF-treated cardiomyocytes fromExample 1 exhibited the calcium clock that is seen in normal SAN cells.

The data in FIG. 4 indicated that a higher incidence of spontaneouslyoscillating calcium transients was seen in SF-treated cardiomyocytes(panel A; SF, n=11; control, n=23; P<0.001; 5 independent experiments),and a faster beating rate of calcium transients was also observed inSF-treated cardiomyocytes (panel B; SF, n=47; control, n=59; P<0.001; 5independent experiments). Panel C and panel D in FIG. 4 arerepresentative diagrams illustrating the calcium transient tracings ofcardiomyocytes in the control group (panel C) and SF-treated group(panel D), respectively. The data in FIG. 4 demonstrated that morespontaneously oscillating calcium transients were seen in cardiomyocytestreated with SF; this result is in line with the underlying theory thatcalcium cycling plays a critical role in the spontaneous beating ofcardiomyocytes.

Further, results from the line-scan confocal imaging (FIG. 5) indicatedthat the LCR preceding each whole-cell Ca²⁺ transient was only observedin SF-treated cardiomyocytes. Specifically, a localized, sub-sarcolemmalCa²⁺ release (LCR) was evidenced by the clustered calcium sparks (whitearrow in FIG. 5) during the diastolic depolarization and before thewhole-cell Ca²⁺ transient. In contrast, for control cardiomyocytes,occasional and randomly distributed Ca²⁺ sparks were only seen after thewhole-calcium Ca²⁺ transient. Moreover, the SF-treated cardiomyocytesexhibited wider and longer-lasting LCRs with higher amplitudes, ascompared with the random Ca²⁺ sparks seen in control cells (FIG. 5).

In sum, these data established that cardiomyocytes treated by SFdisplayed a distinct localized, sub-sarcolemmal Ca²⁺ release profilethat was similar to native SAN pacemakers. The expressions of Ryr2,Cav1.2, Ncx1, Serca2, and PLN did not differ between the control and SFtreated cardiomyocytes (data not shown).

Example 5

HCN4 Inhibitor Suppresses Spontaneous Beating Rate in SF-TreatedCardiomyocytes

The spontaneous beating is tightly regulated by HCN4 channels in nativeSAN pacemakers. Therefore, in this example, cells from Example 1 weretreated with 3 μM ivabradine, a selective HCN4 inhibitor, to evaluatethe impact of HCN4 on the function of SF-treated cardiomyocytes.

As shown in FIG. 6A, ivabradine suppressed the beating rate (i.e., thecycling rates of spontaneous calcium transient) of SF-treatedcardiomyocytes by 57%, as compared with the baseline measurement fromSF-treated cells that were not treated with ivabradine. On the otherhand, the beating rates of control cells treated with ivabradine did notdiffer significantly from the baseline measurement of the control (SF,n=15, P=0.002; control, n=32, P=0.24; 6 independent experiments, Pvalues were determined by paired t test).

FIG. 6B and FIG. 6C are representative diagrams illustrating the calciumtransient tracings of cardiomyocytes in the control group and SF-treatedgroup. Reference in first made to FIG. 6B, which demonstrates that forcontrol NRVMs, the administration of ivabradine did not significantlyalter the rate of calcium transient (bottom panel), as compared with thebaseline measurement (top panel). In contrast, as could be seen in FIG.6C, in the SF treatment group, the rate of calcium transient ofivabradine-treated cardiomyocytes (bottom panel) was significantly lowerthan that of the baseline measurement (top panel).

Example 6

SF Treatment Induces Morphology Conversion of Cardiomyocytes

Each SAN cells had a distinct morphology that is elongated orspindle-like, while cultured ventricular cardiomyocytes often exhibiteda polyhedral central body with cytoplasmic radial projections. As couldbe seen in in panel A of FIG. 7, the control NRVM had a long axisslightly longer than the short axis and four radial projections. On theother hand, the cardiomyocyte treated with SF (from Example 1) had along axis that was 5 times longer than the short axis, which resembledthe morphology of native SAN cells; also, it lacked the radialprojections (see, panel B, FIG. 7).

The dimension of the cells was measured (SF, n=22; control, n=29; 5independent experiments), in which the longest cell length wasconsidered as the long axis, while the axis perpendicular to the longaxis was the short axis. Reference is first made to panel A of FIG. 8,which demonstrated that the lengths of the long axis in both controlcells and SF-treated cells were substantially the same (P=0.78). Thelengths of the short axis in the aforementioned two groups, however,were statistically different, in which the length of short axis inSF-treated cardiomyocytes was about one-third of that in control cells(panel B, FIG. 8; P<0.001). Hence, the long-to-short axis ratio of theSF-treated cardiomyocytes was significantly greater than that of controlcells (panel C, FIG. 8; P<0.001).

These data suggested that the present SF treatment was able to inducethe morphological conversion for NRVMs, and the morphology ofthus-transformed SAN-like cells bore a high resemblance to native SANcells.

Example 7

SF-Induced Automaticity in Cardiomyocytes Responds to AutonomicRegulations

Data form immunostaining analysis demonstrated that muscarinic receptors(M2) and sympathetic receptors (β1) were expressed in SF-treatedcardiomyocytes from Example 1 (see, FIG. 9). In all panels, nuclei werestained with DAPI (blue fluorescence); panel A, green fluorescence:F-actin (a marker for cardiomyocytes); panel B, red fluorescence:muscarinic receptor; panel C: merge image of panels A and B; panel D:green fluorescence: sarcomeric actinin (α-sm, a marker forcardiomyocytes); panel E: red fluorescence: sympathetic β1 receptor;panel F: merge image of panels D and E.

Isoproterenol is a non-selective β-adrenergic receptor agonist, which isused for pharmacological sympathetic stimulation to evaluate thephysiological sympathetic regulation of sinus node. In this example, thefunctional regulations of autonomic neurotransmitters in SF-treatedcardiomyocytes were investigated using the isoproterenol treatment (1μM). The results in panel A of FIG. 10 indicated that the treatment ofisoproterenol accelerated the beating rate of SF-treated cardiomyocytesto 162.4±9.6 bpm, which was faster than the beating rate of controlcells treated isoproterenol (87.6±14.7 bpm), and the differences areregarded as statistical significant (n=10, P=0.001, 5 independentexperiments). Moreover, as could be seen in panel B of FIG. 10, anincrement of 87.1±12.4 bpm was observed between the beating rates ofSF-treated cells without or with the isoproterenol treatment. Theincrement in SF-treated cardiomyocytes was statistically higher thanthose (i.e., 49.2±8.3 bpm) between control cells without and with theisoproterenol treatment (n=10, P=0.02, 5 independent experiments).Panels C to E illustrated the calcium transient tracings ofcardiomyocytes in control (C, E) and SF-treated (D, F) groups without(C, D) or with (E, F) the isoproterenol treatment; the data in thesediagrams indicated that the interval change of beating rate inSF-treated cardiomyocytes was much higher than that of the controlcardiomyocytes.

Overall, these data suggested that the beating rate and interval changeof beating rate of SF-treated cardiomyocytes were regulated bysympathetic stimulation.

Example 8

SF Induces In Situ Transformation of Cardiomyocytes into Pacemaker Cells

30 to 50 μL of SF hydrogel or saline (control) were injected into theleft ventricular apex of rat (male, 10 to 12 weeks old) underthoracotomy. Three weeks later, we ablated the atrioventricular (AV)node and created an AV block under thoracotomy and general anesthesia.Diagrams in FIG. 11A are representative ECG tracing before AV nodeablation (baseline) and after AV node ablation in SF-treated (A and B)and control rats (C and D).

As could be appreciated, after AV node ablation, the electricalconduction from sinus node was blocked, the escaped junctional rhythmjust below the AV node took over the ventricular activation;nonetheless, the rate was very slow (24 bpm) in control rats (FIG. 11A,panel D, lead I). In control rats, the electrical axis remains similarto those conducted from the native AV node—His system; therefore, QRSmorphology of lead I, II, and III is all positive (inferior axis), andno electrical firing was noted over the injection site of the LV apex.On the other hand, for rats receiving the SF hydrogel injection, theescaped rhythm came from the LV apex, the injection site (superior axis,negative QRS voltage over lead II and III, with RBBB pattern) at therate of 120 bpm, and therefore, the axis of electrical conduction wasreversed, as compared with that from the native AVN-His system.

We further performed optical mapping to determine whether the escaperhythm in SF-treated rat came from the injected site. FIG. 11B is anisochrone map illustrating the activation time in left ventricle; ascould be observed in FIG. 11B, the earliest activation site (i.e., theorigin of the escape rhythm) came from the injection site of the SFhydrogel, which located at the left ventricular apex.

A concentrated SF solution containing approximately 12% (w/w) silkfibroin was also injected to the left ventricular apex of rats. Theresults, as provided in FIG. 11C indicated that the injection of the SFsolution also induced the in-situ transformation of non-pacemakercardiomyocytes into pacemaker cells, as several escape rhythms wereobserved.

Together, these findings indicated that the present SF treatment mayinduce the formation of pacemaker cells in situ, and these inducedpeacemaker cell may function as the biological pacemakers to treatbradyarrhythmias when the sinus node or cardiac conduction system isbroken.

Example 9

SF-Treated Cardiomyocytes Express Pacemaker-Specific TranscriptionalFactors

Recent research revealed that during the embryogenesis stage, thesimultaneous activation of Tbx18 and Shox2 plays an important role inthe differentiation of SAN progenitors to mature SAN cells. In thisexample, the expressions of these transcriptional factors in SF-treatedcardiomyocytes from Example 1 were determined. The results, assummarized in FIG. 12, indicated that the expressions of bothtranscriptional factors were increased significantly in SF-treatedcardiomyocytes, as compared with those in control cells (n=7-10,P<0.05).

The upregulation of transcriptional factors such as Tbx18 and Shox2observed herein is in line with the expression profile of SAN cells inthe embryogenesis phase.

Example 10

SF-Treated Cardiomyocytes Exhibit Comparable Beating Rate in Relative toTBX18-Reprogrammed Cardiomyocytes

Adenoviral TBX18 vector (Vector Biolabs (Philadelphia, Pa., USA); HumanTBX18, BC157841) transduction has been used to create biologicalpacemaker in several animal models, including Guinea pigs and pigs. Theadenoviral TBX18 gene therapy has been moving into a phase I clinicalstudy.

The data in FIG. 13 demonstrated that there was no statisticallysignificant difference between the maximal beating rates observed inSF-treated cardiomyocytes and neonatal cardiomyocytes 3 days after thetreatment of TBX18 adenoviral vectors (SF, n=50; TBX18, n=9; P=0.69).Since the maximal beating rates of SF-treated cardiomyocytes andTBX18-reprogrammed cardiomyocytes were comparable, it is believed thatthe present SF-treated cardiomyocytes have a great potential forclinical application as the biological pacemaker.

The experimental data provided in the present disclosure establishedthat non-pacemaker cardiomyocytes could be effectively transformed intocells with pacemaker activity by the treatment of silk fibroin. Thetransformed cells have distinct electrophysiologic characteristics, aswell as morphology characteristics, that are not seen in theirnon-transformed counterparts. Moreover, the gene expression profile oftransformed cells is similar to that of native sinoatrial node pacemakercells, and they undergo similar regulatory mechanisms as the naturalpacemaker cells do. Taken together, the transformed cardiomyocytes arecomparable to natural sinoatrial node pacemaker cells in terms of theirfunction, morphology, gene expressions, and regulatory mechanisms.Accordingly, the silk fibroin-transformed cardiomyocytes are SAN-likecells and may be used to take place of the natural pacemaker cell in thepatients with sinoatrial node damage or other condition associated withcardiac arrhythmias.

It will be understood that the above description of working examples andembodiments is given by way of example only, and that variousmodifications may be made by those with ordinary skill in the art. Theabove specification, examples and data provide a complete description ofthe structure and use of exemplary embodiments of the invention.Although various embodiments of the invention have been described abovewith a certain degree of particularity or with reference to one or moreindividual embodiments, those with ordinary skill in the art could makenumerous alterations to the disclosed embodiments without departing fromthe spirit or scope of this invention.

What is claimed is:
 1. Pacemaker cells, generated using a methodcomprising the step of, culturing a cell population comprisingnon-pacemaker cardiomyocytes with an effective amount of a silk fibroinbiomaterial so that one or more of the non-pacemaker cardiomyocytes aretransformed into the pacemaker cells.
 2. The pacemaker cells of claim 1,wherein the pacemaker cells comprise sinoatrial node (SAN)-like cells.3. The pacemaker cells of claim 2, wherein the SAN-like cells exhibit anincreased expression level of hyperpolarization-activated, cyclicnucleotide-gated potassium channel 4 (HCN4) gene or connexin 45 gene, orboth, compared with the non-pacemaker cardiomyocytes.
 4. The pacemakercells of claim 2, wherein the SAN-like cells exhibit sinoatrialnode-specific calcium clock.
 5. The pacemaker cells of claim 1, whereinthe silk fibroin biomaterial is a silk fibroin solution, a silk fibroinparticle, a non-woven silk fibroin mat, a silk fibroin hydrogel, a silkfibroin film, or a silk fibroin scaffold.
 6. The pacemaker cells ofclaim 5, wherein the silk fibroin film comprises, a biodegradablepolymeric film having a plurality of amines group on the surfacethereof; and a plurality of silk fibroins crosslinked with thebiodegradable polymeric film via the plurality of amine groups.
 7. Apharmaceutical composition for treating cardiac arrhythmia, comprisingan effective number of pacemaker cells of claim 1 and a pharmaceuticallyacceptable carrier thereof.
 8. A method for generating pacemaker cells,comprising the step of contacting a cell population comprisingnon-pacemaker cardiomyocytes with an effective amount of a silk fibroinbiomaterial so that one or more of the non-pacemaker cardiomyocytes aretransformed into the pacemaker cells.
 9. The method of claim 8, whereinthe cell population and the silk fibroin biomaterial are contacted invitro.
 10. The method of claim 8, wherein the cell population and thesilk fibroin biomaterial are contacted in vivo.
 11. The method of claim8, wherein the silk fibroin biomaterial is a silk fibroin solution, asilk fibroin particle, a non-woven silk fibroin mat, a silk fibroinhydrogel, a silk fibroin film, or a silk fibroin scaffold.
 12. Themethod of claim 11, wherein the silk fibroin film comprises, abiodegradable polymeric film having a plurality of amines group on thesurface thereof; and a plurality of silk fibroins crosslinked with thebiodegradable polymeric film via the plurality of amine groups.
 13. Amethod for treating a subject suffering from cardiac arrhythmia,comprising the step of administering to the subject an effective numberof pacemaker cells of claim
 1. 14. The method of claim 13, wherein thepacemaker cells are generated from non-pacemaker cardiomyocytes derivedfrom the subject.
 15. The method of claim 13, wherein the silk fibroinbiomaterial is a silk fibroin solution, a silk fibroin particle, anon-woven silk fibroin mat, a silk fibroin hydrogel, a silk fibroinfilm, or a silk fibroin scaffold.
 16. The method of claim 13, whereinthe silk fibroin film comprises a biodegradable polymeric film having aplurality of amines group on the surface thereof, and a plurality ofsilk fibroins crosslinked with the biodegradable polymeric film via theplurality of amine groups.
 17. A method of treating a subject sufferingfrom cardiac arrhythmia, comprising the step of administering to theheart of the subject an effective amount of a silk fibroin biomaterial.18. The method of claim 17, wherein the silk fibroin biomaterial is asilk fibroin solution, a silk fibroin particle, a non-woven silk fibroinmat, a silk fibroin hydrogel, a silk fibroin film, or a silk fibroinscaffold.
 19. The method of claim 18, wherein the silk fibroin filmcomprises a biodegradable polymeric film having a plurality of aminesgroup on the surface thereof, and a plurality of silk fibroinscrosslinked with the biodegradable polymeric film via the plurality ofamine groups.